Alzheimer's disease (AD) is characterized by hallmark lesions such as amyloid rich senile plaques and neurofibrillary tangles. Amyloid beta (Abeta) peptides, which are derived from the proteolytic processing of one or more of the alternatively spliced Abeta precursor protein (APP) isoforms are the building blocks of the amyloid fibrils in the neuritic plaques that accumulate in the brains of patients with AD. The 2 major forms of Abeta in amyloid plaques terminate at amino acid 40 or 42/43 of this peptide, and brain cells secrete much high levels of Abeta/1-40 than Abeta/1-42. However, Abeta/1-42 is much less soluble than it aggregates far more readily than Abeta/1-40. Indeed, Abeta/1-42 predominates in the amyloid plaques that riddle the AD brain. Thus, the elucidation of intracellular pathways that lead to the production of Abeta particularly Abeta/1-42 in brain cells such as the neuron would increase our understanding of those mechanisms that underlie the pathogenesis of senile plaques in AD. During the last funding cycle of this Project, we identified the endoplasmic reticulum/intermediate compartment (ER/IC) as a novel site for the production of Abeta/1-42 but not Abeta/1-40 in human NT2N neurons. Significantly, some of the ER/IC produced Abeta/1-42 formed a stable, insoluble pool of intracellular Abeta that progressively accumulated as the NT2N neurons aged in culture. This aggregated intracellular Abeta/1-42 could compromise the survival of selectively vulnerable neurons. Thus, the studies proposed in this renewal application will test the hypothesis that perturbation of APP processing leading to progressive intracellular accumulation of Abeta particularly Abeta/1-42 in AD brains may lead to neuron death. To accomplish this, we will use reagents and tools that we have developed during the last funding cycle to dissect the different intracellular pathways that produce Abeta/1-40 and Abeta/1-42. We will determine whether or not Abeta/1-42, soluble intracellular Abeta/1-40 and Abeta/1-42 as well as insoluble, aggregated Abeta/1-40 and Abeta/1-42. Close collaboration with other investigators in the Program Project will allow us to determine how the production of the different pools of Abeta particularly intracellular Abeta/1-42 are regulated. These studies should also provide clues to the importance of intracellular Abeta in neuron death.